Post-translational modification by ubiquitin and ubiquitin-like proteins (ublps) is a predominant cellular regulatory mechanism. Ubiquitination regulates a vast array of biological processes, including cell division, the immune response, and embryonic development. As a result, defects in the ubiquitin pathway are associated with numerous diseases, particularly cancer, and disorders associated with aging, such as neurodegenerative disorders and arid muscle wasting. In addition to ubiquitin, over 10 ublps have been found in higher eukaryotes. Ublps have structures and sequences that closely resemble ubiquitin, but they direct their targets to distinct destinies. Therefore, it is important to understand the molecular bases that define the specificity of the ubiquitination process. Our long-term goal is to understand how ubiquitin and ublps are directed to their particular targets to control processes involved in diseases such as cancers and neurodegenerative disorders. Ubiquitin is ligated to targets by a cascade involving a series of three enzymes in classes known as E1, E2, and E3. Despite the importance of these enzymes, little is known how ubiquitin is selected by ubiquitinating enzymes. We hypothesize that the specificity of ubiquitinating enzymes is dictated by a combination of positive selection for interacting with ubiquitin, and negative selection against the wrong ublp. We are focusing on the E1, E2 and E3 enzymes involved in cell proliferation and involved in regulating tumor suppressor proteins. Because these enzymes are important for cell proliferation, and play roles in pathways that lead to cancers, these enzymes may serve as good targets for anti-mitogenic agents. The recent approval of the proteasome inhibitor Bortezomib (VelcadeTM) for treatment of multiple myeloma underscores the therapeutic potential for targeting enzymes in the ubiquitin, and ublp, pathways, and highlights the importance of understanding the detailed mechanisms and specificities of these enzymes. We plan a multidisciplinary approach that combines biochemical, enzymological and structural analysis of ubiquitinating enzymes, enzymes in ublp conjugation cascades, mutants and complexes, in order to understand the specificity of ubiquitination.